Recently, layer-by-layer (LbL) assembled multilayer nanofilm has been widely studied for various application. The weak polyelectrolytes such as poly(acrylic acid) (PAA), poly(allylamine hydrochloride) (PAH), poly-L-lysine (PLL), and polyethylenimine (PEI) are suitable candidates for the LbL assembly. Even though many research groups have already reported the various method to tailor nanoscopic features of multilayer film, there are still some limitations. Here, we report a noble CO2 bubble method to precisely modify the multilayer film during LbL assembly. The difference in thickness, surface morphology, and mass between pristine LbL film and CO2-treated film were analyzed by profilometry, atomic force microscopy, scanning electron microscopy, quartz crystal microbalance. The internal structure of the films was explored by analyzing the release of incorporated doxorubicin (DOX) in each film by photoluminescence.